NKCC1 Is Phosphorylated in Rat Inner Medullary Collecting Duct (IMCD)

Abstract

Previously, we demonstrated a bumetanide‐inhibitable increase in IMCD cell height in response to vasopressin (AVP) in isolated perfused tubules in which the bath osmolality was increased to 490 mOsm (lumen 290 mOsm). This suggests that AVP induced cell swelling was not due to apical water entry, but rather basolateral solute uptake via NKCC1, followed by water entry. To address the role of NKCC1 we used an N‐terminal directed antibody recognizing total NKCC1 (α‐NT) and a phospho‐directed antibody targeted to Thr‐212 and Thr‐217 in the N‐terminal tail of human NKCC1 (R5). Using the α‐NT antibody for immunofluorescence, we confirmed that NKCC1 is localized to the basolateral membrane of the rat IMCD cell. To test if NKCC1 is phosphorylated, we used the R5 phospho‐antibody in immunoblotting. The antibody recognized 170 and 150 kDa bands in IMCD suspensions under control conditions (290 mOsm). Hypertonicity (490 mOsm) caused the band density of the lower band to increase by 2‐fold (P<0.05, n=3). There was no obvious change in mobility of proteins recognized by a COOH‐tail antibody. A dephosphorylation experiment using λ‐phosphatase confirmed that the R5 antibody recognized only phosphorylated proteins. AVP alone did not change the band pattern with the R5 antibody. We conclude that in IMCD cells NKCC1 is a phospho‐protein and that hypertonicity affects the mobility or phosphorylation state of the NKCC1 phosphoprotein.